Joint transfer of genetic markers in Bacillus subtilis

Takahashi, I. (McMaster University, Hamilton, Ontario, Canada). Joint transfer of genetic markers in Bacillus subtilis. J. Bacteriol. 91:101-105. 1966.-To compare the processes of genetic incorporation in transduction and transformation in Bacillus subtilis, several groups of linked markers were selected and the degree of linkage was determined by the two means of genetic exchange. Bacteriophage PBS 1 was used in transduction experiments. In all cases, frequencies of joint transfer, as expressed by the cotransfer index or by percentage of joint transfer, were higher in transduction than in transformation. With a pair of closely linked markers, the frequency of joint transduction was only slightly higher than that of joint transformation. On the other hand, a considerably higher degree of linkage was obtained by transduction when loosely linked markers were examined. It appears that the size of donor chromosome transferred by transducing phage particles is much larger than that incorporated by recipient cells in transformation. It is suggested that transduction in B. subtilis may be a useful tool in extending further the linkage groups established by the transformation technique.     

Transformation and Transduction in Recombination-defective Mutants of Bacillus subtilis

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.     

Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.     

Genetic mapping of the bacitracin synthetase gene(s) in Bacillus licheniformis

The map position of a mutation in the bacitracin synthetase gene(s) in Bacillus licheniformis ATCC 10716 was determined by transduction with phage SP-15. Results indicate that it is linked to the lys and trp loci and is distinct from the known sporulation loci on the chromosome of Bacillus licheniformis. The defect(s) of the enzyme complex were analysed in terms of its ability to bind covalently 14C-labelled amino acid precursors of the bacitracin molecule.     

Genetic mapping of a bacteriophage resistance mutation, pha-1, and the thi-1 mutation in Bacillus licheniformis.

A mutation, designated pha-1, resulting in resistance to bacteriophage LP-52, mapped between gly-3 and pepA1 in Bacillus licheniformis by transduction with phage SP-15. The thi-1 mutation was also found to be linked to gly-3. The map order was thi-1--gly-3--pha-1--pepA1--his-9.     

Growth and Cultivation of the Unusual Generalized Transducing Bacillus Bacteriophage SP-15

Additional properties of SP-15, a generalized transducing bacteriophage notable for the ability to transfer an unusually large fragment of deoxyribonucleic acid (DNA) to Bacillus subtilis and B. licheniformis, are presented together with improved methods that enhance its utility. Simple means have been found to provide the rigid control over moisture that is necessary for the assay of plaque-forming units (PFU). Reproducible procedures for propagating transducing phage, which depend upon an appropriate mixing of PFU with uninfected bacteria, have replaced less reliable methods that utilized infected spores. Transduction of B. subtilis W-23 increased linearly when MgSO(4) in recipient cell-SP-15 mixtures was increased from 0.005 to 0.03 m. Methods have been developed that protect SP-15 from the damaging effects of CsCl and of osmotic shock subsequent to dilution. Evidence that the PFU and transducing particles of lysates decay at the same slow rate during extended storage suggests that the decay is a result of damage to protein rather than to DNA. One-step growth experiments, in which SP-15 was propagated on B. subtilis W-23-S(r)/1 mg, indicated a latent period of 100 min, a rise period of 60 min, and a burst size of 25 to 34 PFU per infected cell. These findings suggest explanations for some of the technical difficulties SP-15 has presented.     

Sequential Replication of the Chromosome of Bacillus licheniformis

A sequential replication map of the chromosome of Bacillus licheniformis was constructed by employing the method of gene-frequency analysis presented by Yoshikawa and Sueoka. Our analysis of 11 genetic markers was based on the hypothesis that the chromosome initiated replication at a fixed origin and proceeded in a linear fashion to the terminus. The proposed locations of markers were validated by cotransformation and cotransduction analyses. Bacteriophage SP-15 cotransduced markers that failed to show linkage by transformation.     

Citric acid cycle: gene-enzyme relationships in Bacillus subtilis

The genetic location of mutations affecting the citric acid cycle and the properties of mutants of Bacillus subtilis possessing these mutations have been examined. Genes coding for the component enzymes of the cycle were found to be unlinked to each other and thus do not form an operon. The mutational defect in a mutant lacking fumarase mapped between thr-5 and cysB3. Mutations causing inability to produce isocitrate dehydrogenase and succinate dehydrogenase were found to map between argA11 and leu-1. The alpha-ketoglutarate dehydrogenase mutations were mapped at the terminal end of the B. subtilis chromosome through a weak linkage in phage PBS-1 transduction of one class of these mutations of ilvA2 and metB4. A second class of alpha-ketoglutarate dehydrogenase mutations mapped closer to ilvA2 and metB4 but still terminal with respect to these markers. Aconitaseless mutants possessed mutations that could not be linked to any of the known transducing segments of the chromosome. An effect of mutation conferring loss of one enzyme of the cycle on the specific activity of the other enzymes in the cycle was observed.     

Mapping of the gene for endo-β-1,3-1,4-glucanase of Bacillus subtilis

Abstract An integrating plasmid has been used to mutagenise the gene coding for endo-β-1,3-1,4-glucanase of Bacillus subtilis . The gene, named bgl , has been mapped by PBS-1 transduction to the sacA-pureA region of the B. subtilis chromosome and is closely linked to the hutP 1 locus. The order of markers in this region is sacA 321- thiC 5- bgl - hutP 1- purA 16.     

Some Properties of the PBP1 Transduction System in Bacillus pumilus

Bacteriophage PBP1 is a flagella-specific virus that performs generalized transduction in strains of Bacillus pumilus. PBP1 is morphologically and serologically distinct from two other flagella-specific phages, PBS1 and SP-15, which perform generalized transduction in certain Bacillus species. The DNA extracted from PBP1 particles has a buoyant density of 1.690 g/cm(3) in cesium chloride gradients, a melting temperature of 86.1 C, and a sedimentation velocity of 47S in neutral sucrose gradients. Assuming the molecule is a linear duplex, PBP1 DNA has a molecular weight of approximately 76 x 10(6). In two strains of B. pumilus which are sensitive to both PBP1 and PBS1, co-transducible genetic markers are more tightly linked by PBS1 transduction than by PBP1 transduction. The size of the fragment of bacterial DNA carried by PBP1-transducing particles, inferred from transduction studies and sedimentation analysis of viral DNA, suggests that PBP1 may be useful for genetic studies of extrachromosomal DNA elements present in two strains of B. pumilus. Genetic exchange of chromosomally located genes between the plasmid(+) and plasmid(-)B. pumilus strains NRS 576 and NRRL B-3275 has been demonstrated by PBP1 transduction.     

Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome.

A revision of the linkage map of the Bacillus subtilis 168 chromosome has been undertaken with the use of the generalized transducing phage PBS1. The mapping of four new markers (narB1, mtlB1, aroI906, and tre-12) has allowed a determination of the relative orientation of the purB-dal segment and its linkage with the lin markers. The chromosomal segment comprised between the sacQ36 and gtaA12 markers has been linked with the narA1, ctrA1, and sacA321 markers. The recA1 marker has been mapped relative to the thyA and citB17 markers. Indications of linkage have been found between the tre-12 and catA markers and the aroG932 and sacQ36 markers. According to these results, a circular genetic map of the chromosome of B. subtilis 168 is presented. Taken together, the transduction data and the order of marker replication determined by Harford in the accompanying paper support strongly the hypothesis of a symmetrical and fully bidirectional mode of replication for the B. subtilis 168 chromosome.     

Genetic Mapping of Genes Concerned with Glutamyl Polypeptide Production by Bacillus licheniformis and a Study of Their Relationship to the Development of Competence for Transformation

By transduction with SP-15, we have mapped some of the genetic sites involved in glutamyl polypeptide (Pep) production by Bacillus licheniformis 9945A. Eleven peptide mutations were assigned to group 1 which lies between the gly-42 and his-6 markers. Three other mutations were assigned to group 2 which lies between the ade-2 and str-1 sites. A series of 28 additional Pep(-) mutants did not belong to either group 1 or 2; the chromosomal location of those peptide mutations is unknown. All three mutants of group 2 were highly transformable, but only two of group 1 transformed well under the conditions employed. Knowing the chromosomal markers that were linked to peptide mutations made it possible to investigate the effect peptide mutations might have on the development of competence for transformation. Nontransformable organisms were rendered transformable upon the introduction, by transduction, of Pep(-) mutations from transformable strains. These results supported the conclusion that the ability of cells to develop competence for transformation when grown under appropriate conditions was related to the physiological effects of particular peptide mutations.     

Genetic Mapping of a Defective Bacteriophage on the Chromosome of Bacillus subtilis 168

A genetic marker responsible for the killing activity of PBSX, a defective phage carried by Bacillus subtilis 168, has been located on the bacterial chromosome. Two mutant strains of B. subtilis 168, which produced tailless phage particles upon mitomycin C induction, were shown to carry lesions, designated xtl-1 and xtl-2, which were linked by transformation and PBS1-mediated transduction to metC. The link-age relationship between xtl and adjacent auxotrophic markers was determined by three-factor PBS1 transduction, the suggested order of markers being argO 1 metA metC xtl.     

Productive Infection of Bacillus subtilis 168, with Bacteriophage SP-10, Dependent upon Inducing Treatments

Strains of Bacillus that harbor defective phage PBSX were found to be insensitive to SP-10(C), although the phage adsorbed to these insensitive strains. Strains that did not carry the phage were sensitive to SP-10(C). B. subtilis 168 ind(-), which can be tranduced by SP-10(C) but is nonpermissive for the phage, was rendered phage-sensitive after treatment with ultraviolet (UV) light or mitomycin C. After induction with UV light, maximal sensitivity to SP-10(C) was obtained at a multiplicity of infection (MOI) of approximately 14; with mitomycin C induction, an MOI of approximately 1.0 was required. Phage maturation in sensitized cells was followed by plating infected streptomycin-sensitive cells in the presence of streptomycin at various stages during phase development. The latent period was estimated at 60 to 75 min. We suggest that the resistance of B. subtilis 168 to SP-10 is controlled, at least in part, by the presence of a defective prophage.     

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.     

Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10

Taylor, Martha J. (Fort Detrick, Frederick, Md.), and Curtis B. Thorne. Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10. J. Bacteriol. 91:81-88. 1966.-Spores of Bacillus subtilis W-23-S(r) infected with transducing phage SP-10 served as convenient inocula for broth cultures from which transducing phage was harvested. Methods are described for producing highly infected spores. The inoculum level of infected spores in nutrient broth-yeast extract-glucose medium affected the transducing efficiency of SP-10 in lysates of these cultures. Phage in lysates of cultures inoculated with about 10(5) or fewer spores per milliliter transduced 20- to 350-fold more efficiently than did phage in lysates from cultures inoculated with 10(6) to 10(7) spores per milliliter. Transduction frequencies in the order of 10(-5) per plaque-forming unit were obtained routinely, and some infected-spore preparations yielded phage that gave frequencies as high as 10(-4). The combination of inoculum level and incubation time required to produce the best transducing phage had to be determined empirically for each batch of infected spores. Several possible explanations for the difference between lysates having high (HTE) and those having low (LTE) transducing efficiency were ruled out by special experiments. The hypothesis is presented that some cultural condition resulting from a relatively low inoculum of phage-infected spores favors the incorporation by phage particles of bacterial deoxyribonucleic acid (DNA) in the manner required for the production of transducing phage. Support for this hypothesis is a demonstration, through transformation experiments with DNA extracted from HTE and LTE phage particles, that populations of HTE phage particles yielded significantly more (7 to 27 times) transforming activity per microgram of DNA than did populations of LTE phage.     

地衣芽孢杆菌原生质体的制备、再生及转化研究

目的：提高地衣芽孢杆菌原生质体的产量和形成率,为进一步提高原生质体转化率打下基础。方法：通过酶解法对地衣芽孢杆菌工业生产菌株Bacillus licheniformis303原生质体的制备及再生条件进行了研究。考察了菌体生长状态、溶菌酶浓度、处理时间、渗透压稳定剂和再生培养基等因素对地衣芽孢杆菌原生质体的制备及再生的影响。结果：对数生长后期的菌体,以SMMP作渗透压稳定剂,溶菌酶浓度为100mg/mL,37℃下酶解30min,原生质体生成量可达8×109个/mL;再生培养基选用含1mol/L琥珀酸钠的DM3时,再生率最高可达17%。在此条件下,采用PEG法将游离型质粒pHY-P43-secQ转化宿主菌B.lichenifor-mis303,转化率可达10~15 CFU/μg DNA。     

Structure and glycosylation of lipoteichoic acids in Bacillus strains.

The occurrence, structure, and glycosylation of lipoteichoic acids were studied in 15 Bacillus strains, including Bacillus cereus (4 strains), Bacillus subtilis (5 strains), Bacillus licheniformis (1 strain), Bacillus polymyxa (2 strains), and Bacillus circulans (3 strains). Whereas in the cells of B. polymyxa and B. circulans neither lipoteichoic acid nor related amphipathic polymer could be detected, the cells of other Bacillus strains were shown to contain lipoteichoic acids built up of poly(glycerol phosphate) backbone chains and hydrophobic anchors [gentiobiosyl(beta 1----1/3)diacylglycerol or monoacylglycerol]. The lipoteichoic acid chains of the B. licheniformis strain and three of the B. subtilis strains had N-acetylglucosamine side branches, but those of the B. cereus strains and the remaining two B. subtilis strains did not. The membranes of the B. licheniformis strain and the first three B. subtilis strains exhibited enzyme activities for the synthesis of beta-N-acetylglucosamine-P-polyprenol and for the transfer of N-acetylglucosamine from this glycolipid to endogenous acceptors presumed to be lipoteichoic acid precursors. In contrast, the membranes of the other strains lacked both or either of these two enzyme activities. The correlation between the occurrence of N-acetylglucosamine-linked lipoteichoic acids and the distribution of these enzymes is consistent with the previously proposed function of beta-N-acetylglucosamine-P-polyprenol as a glycosyl donor in the introduction of alpha-N-acetylglucosamine branches to lipoteichoic acid backbone chains.     

Thermotolerant varieties of Bacillus licheniformis isolated from desert environments

One hundred and nineteen thermotolerant and thermophilic Bacillus strains isolated from solar-heated and non-heated environments in Jordan were classified by numerical techniques. Some strains were classified into thermophilic taxa which did not equate with established species. However, most of the isolates were identified phenotypically as Bacillus licheniformis, a conclusion supported by the high DNA hybridization which was detected between these strains and a reference strain of this species (gt64% at optimal renaturation temperature). Several of the B. licheniformis isolates had a higher ratio of iso-C₁₅ and iso-C₁₇ fatty acids to the anteiso equivalents in their membranes than the reference strain of B. licheniformis and they grew more strongly at high temperature than the reference strain. This suggests that the B. licheniformis isolates represent thermotolerant variants of this species.     

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.